JPH0716440B2 - Glucose measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring glucose. More specifically, the present invention applies reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) and aldose reductase to a glucose-containing sample to reduce glucose. NADH or NA conjugated with
This is a method of measuring the amount of NADH or NADPH reduced by the oxidation reaction of DPH and then measuring the amount of glucose in the sample.

Blood glucose is the most important source of energy in the body, and its concentration depends on the absorption of sugar from the intestinal tract, gluconeogenesis and glucogen synthesis and decomposition in the liver, sugar utilization in peripheral tissues,
It depends on various factors such as excretion from the kidney, and its regulation is closely related to autonomic nerves and various hormones.

Insulin is involved in lowering blood glucose, and epinephrine, glucagon, growth hormone, adrenocortical hormone, thyroid hormone, etc. are involved in increasing blood glucose, and the blood glucose level is delicately regulated by their antagonism and cooperative action.

Clinically, it is known that glucose levels fluctuate during endocrine diseases, functional disorders of the pancreas, liver, kidney, etc.,
Measuring glucose levels in serum and urine will have important implications for their impairment and prognostic diagnosis.

As a conventional chemical method for blood glucose measurement, the reduction method, which is a color reaction utilizing the reducing action of glucose in an alkaline solution, and the fact that glucose reacts with an aromatic amine under strong acidity to cause condensation coloring There is a condensation method. The reduction method has the drawback that it is strongly influenced by non-sugar reducing substances such as ascorbic acid, uric acid, glutathione, and glucuronic acid, and that glucose cannot be accurately measured, and that the condensation method is simple but has low specificity. is there.

As the enzyme method, an enzyme electrode method using glucose oxidase, a method of measuring oxidative coloration in the coexistence of peroxidase (GOD-POD method), and glucose-6-phosphate dehydrogenase coupling with hexokinase are used. (HK-G6PD
Method) or using only glucose dehydrogenase.
There is a method of measuring the reaction production amount of DH or NADPH by the absorbance at 340 nm. However, although the enzyme electrode method is excellent in rapidity, it has a drawback that it is affected by coexisting substances. In addition, since the GOD-POD method is an end point measurement, when the glucose concentration becomes high, oxygen is insufficient and the reversal phenomenon of generation occurs.
The HK-G6PD method is an excellent method in terms of accuracy, but it requires a long measuring time because it is an end point measuring method. Although the glucose dehydrogenase method is satisfactory in many respects, it has a drawback that the measurement interval is small. Therefore, with the rapid spread of automatic analyzers in the clinical examination field in recent years, there has been a demand for a glucose quantification method by a reaction rate method with a wide measurement range in a short time.

Generally, when quantifying a substance serving as a substrate of an enzyme based on the reaction rate of the enzyme, it is an essential condition that the Km value for the substrate of the enzyme is sufficiently higher than the substrate concentration in the measurement system.

The present inventors, as a result of intensive research for an effective quantification method of glucose by the reaction rate method, found that the aldose reductase has a very high Km value for glucose of several hundred mM, and further, the glucose reaction rate method. The inventors have found that it is extremely effective to use aldose reductase as a quantification method in, and have completed the present invention.

The present invention allows reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate and aldose reductase to act on a sample containing glucose to reduce reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine. A method for measuring glucose, which comprises measuring the rate of decrease of dinucleotide phosphate.

When practicing the present invention, a sample of NADH or NADPH is used.
In the presence of aldose, aldose reductase is allowed to act in a neutral to weakly alkaline buffer solution, and the oxidative reaction of NADH or NADPH that simultaneously proceeds with the glucose reduction reaction that proceeds by aldose reductase activity is absorbed by ultraviolet absorption (340n
Glucose in the sample can be quantified by measuring the decreasing rate at m).

Sources of aldose reductase used in the present invention include animals and microorganisms, and any aldose reductase may be used. As the buffer solution, triethanolamine, tris, phosphoric acid, good buffer, etc. can be used,
A pH of 6-9 is preferred. The present invention is not limited to the source of aldose reductase, the type of buffer used and the pH.

Since the reaction system of the method of the present invention is simpler than that of the conventional method, there are few error factors, and the influence of substances in the specimen sample can be almost ignored. Even in the measurement of a specimen containing glucose, it is possible to perform accurate and highly sensitive measurement in a short time without diluting the sample, and it is a very useful method in the measurement by the analyzer itself.

Examples of the present invention will be shown below.

Example 1 As a reaction solution, 0.35 u / ml of aldose reductase and
100 mM potassium phosphate buffer (pH 7.0) containing 0.25 mM NADPH
Was placed in a 2 ml cuvette and set in a spectrophotometer whose temperature was adjusted to 37 ° C., 20 μl of standard solutions each containing 0 to 10 g / dl glucose were added, and the change in absorbance per minute at 340 nm was measured. . The results are shown in FIG. From this result, it was confirmed that the solution has linearity up to a solution containing 10 g / dl of glucose and is extremely effective as a reaction rate method.

Example 2 As a reaction solution, about 2 u / ml of aldose reductase and 0.25 m
2 ml of 100 mM potassium phosphate buffer (pH 7.0) containing M NADPH
It was placed in a cuvette and set in a spectrophotometer whose temperature was adjusted to 37 ° C., 20 μm of serum containing 0 to about 5,000 mg / dl of glucose was added to each, and the change in absorbance at 340 nm per minute was measured. The measurement results are shown in Table 1 below.

[Brief description of drawings]

FIG. 1 is a diagram showing the linearity when measuring glucose in Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Arch Biochem Biophys, 238 (2), 1985, p. 670-679 Chem Pharm Bull (Tokyo), 31 (7), 1983, P.P. 2395-2403 Maruo and Tamiya, "Enzyme Handbook" Asakura Shoten (1982-12-1) P. 333-335

Claims (1)

[Claims] 1. A reduced nicotinamide adenine dinucleotide is allowed to act on a sample containing glucose (excluding a glucose single solution) with reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate and aldose reductase. A method for selectively measuring only glucose, which comprises measuring the rate of reduction of leotide or reduced nicotinamide adenine dinucleotide phosphate.